How dysfunctional glial cells lead to abnormal pain signaling in the spinal dorsal horn in neuropathic pain remains a mystery. Activation of glutamate receptors by glutamate is a key step for acute pain transmission and activation of signal transduction pathways leading to initiation and maintenance of pathological pain. Activation of glutamate receptors is governed by three essential factors: the amount of synaptically released glutamate, the rate at which glutamate is removed by glutamate transporters (GTs) and the properties of postsynaptic glutamate receptors. Modern pain treatments directed at the transmission of pain singals have mainly focused either on the presynaptic levels to block glutamate release from presynaptic terminals or on the postsynaptic levels to block glutamate receptors and glutamate activated signal transduction pathways in postsynaptic neurons. Targeting at GTs to correct abnormal activation of glutamate receptors has been much less investigated. One key role for glial cells in regulating signal transmission at the glutamatergic synapse is the removal of synaptic glutamate by glial GTs. Studies on the forebrain have demonstrated that glial GT accounts for more than 94% of all CNS synaptic glutamate uptake and is a key machinery regulating synaptic signal transmission and plasticity. However, the role of glial GTs in regulating synaptic transmission in the spinal dorsal horn and their contribution to abnormal pain signaling have not been established. Based on our preliminary findings, we hypothesize that activation of glial cells induced by nerve injury results in dysfunction of glial GTs, which leads to abnormal activation of ionotropic glutamate receptors in the spinal dorsal horn and behavioral hypersensitivity. This hypothesis will be tested with three specific aims. Specific Aim 1 will determine the relationships between glial GT expression and development of behavioral hypersensitivity, glial activation and release of pro-inflammatory cytokines induced by nerve injury. Specific Aim 2 will determine the contribution of deficient glutamate uptake by glial GTs to abnormal activation of AMPA and NMDA receptors in the spinal dorsal horn and behavioral hypersensitivity in neuropathic rats. Specific Aim 3 will determine the contribution of recovered glutamate uptake by glial GTs to the normalized activation of ionotropic glutamate receptors in the spinal dorsal horn and to the normalized nociceptive behaviors in neuropathic rats after treatments of glial inhibitors or antagonists for pro-inflammatory cytokines. Experiments will be conducted in control (normal naove and sham operated) rats and neuropathic rats induced by partial sciatic nerve ligation, which is known to mimic neuropathic pain induced by partial nerve injury in patients. Multi-disciplinary- techniques, including visualized whole cell voltage clamp recordings from spinal slices, pharmacology, immunohistochemistry and behavioral tests will be applied to test our hypothesis. The proposed study will provide new insights into the synaptic and molecular mechanisms underlying glial-cytokine-neuronal interactions in neuropathic pain and may potentially lead to the use of GTs as a new target for reducing persistent and abnormal activation of glutamate receptors in the management of chronic pain. PUBLIC HEALTH RELEVANCE: How dysfunctional glial cells lead to abnormal pain signaling in the spinal dorsal horn in neuropathic pain remains a mystery. Activation of glutamate receptors by glutamate is a key step for acute pain transmission and activation of signal transduction pathways leading to initiation and maintenance of pathological pain. We hypothesize that activation of glial cells induced by nerve injury results in dysfunction of glial GTs, which leads to abnormal activation of ionotropic glutamate receptors in the spinal dorsal horn and behavioral hypersensitivity. 1